PROJECT SUMMARY/ABSTRACT Until recently, tumor evolution was thought to follow a model in which tumor development results from the stepwise accumulation of mutations conferring cancer cells evolutionary fitness advantages required for unregulated growth and metastasis. Recent groundbreaking work demonstrates that, in fact, it is mutations occurring very early during (or even preceding) neoplastic transformation that dictate tumor evolution, and hence some tumors are ?born to be bad?. These novel findings further suggest that the potential of a tumor to become malignant is under the influence of (epi)genetic/signaling deregulation whose identification may allow for the distinction between malignant and benign lesions. However, the (epi)genetic/signaling alterations driving susceptibility to neoplastic transformation are poorly understood. Compelling work has demonstrated in several organs, including the intestine, that the cancer cell of origin corresponds to a resident multipotent stem cell that has been (epi)genetically altered. Therefore, identifying (epi)genetic/signaling alterations driving neoplastic transformation in resident intestinal stem cells (ISC) offers the possibility to define specific markers and pathways that may be of interest to allow for early detection of tumor initiation and for new targeted therapies against tumor formation. Previous work from our group has identified protocadherin 8 (PCDH8) as specifically enriched in ISC as compared to all other intestinal epithelial cell types. Preliminary work further demonstrates that PCDH8 is markedly decreased in colon cancers and as early as in polyps, as a result of promoter hypermethylation. We also have evidence that PCDH8 forced expression dramatically limits colon cancer cell ability to form tumors in vitro and restricts tumor growth in vivo. Altogether our data indicate that PCDH8 is an ISC marker that functions as a tumor suppressor gene. However, the implication of loss of PCDH8 in ISC neoplastic transformation remains to be demonstrated. Therefore, the proposed studies will test the hypothesis that loss of PCDH8 drives and is required for ISC neoplastic transformation in 2 specific aims. First, we will demonstrate that loss of PCDH8 induces (or at least stimulates) ISC neoplastic transformation and tumor formation. To do this, we will assess if loss of PCDH8 alone or combined with constitutive activation of the Wnt/?-catenin target gene program induces ISC neoplastic transformation in vitro. Using an ISC-specific PCDH8 knock out mouse model with intact or overactivated Wnt/?-catenin pathway, we will also test whether loss of PCDH8 triggers or at least increases tumor formation in vivo. Then we will show that ISC neoplastic transformation involves loss of PCDH8. To do this, we will demonstrate that colon tumors originate from PCDH8-expressing cells but have lost PCDH8 expression using lineage tracing. We will also show that ISC neoplastic transformation can be rescued/reversed by forcing PCDH8 expression in cancer-initiating cells. We believe that defining the role of PCDH8 and associated molecular pathways and cellular functions in intestinal health and cancer will allow for the identification of novel markers and/or targets to improve early colon cancer detection and treatment.